Manganese Superoxide Dismutase (MnSOD) is a component of the cellular antioxidant defense mechanism that is necessary for mitochondrial function, cellular energy production and cell viability. Native MnSOD is a mitochondrial protein that is imported from the cytoplasm and localized to the mitochondrial matrix, where it scavenges superoxide free radicals or anions and converts these reactive oxygen species into the benign oxidant, hydrogen peroxide (H2O2), and oxygen. MnSOD is expressed in all cell types and provides an essential function. MnSOD importance was clearly illustrated in mice neonatal offspring containing a homozygous disruption (knockout) of MnSOD gene. These mice develop multisystem, mitochondrial energy-loss pathologies that include cardiomyopathy, neurological and liver dysfunction and exhibit perinatal lethality. The homozygous MnSOD knock-out serves as a proof-of-principle for complete loss of MnSOD function.
The multisystem energy-loss phenotype in the MnSOD knockout mouse may be due to an adverse accumulation of superoxide free radicals within the mitochondria upon the onset of an aerobic environment, causing loss of the ATP-synthesizing capacity of mitochondria and either initiating premature cell death by necrosis or by initiating the mitochondrial membrane permeability transition, causing release of mitochondrial pro-apoptotic proteins from the mitochondria due to depolarization of the mitochondrial membrane. Adverse accumulation of superoxide will result in spontaneous dismutation of superoxide into hydrogen peroxide, which is normally a benign oxidant unless in the presence of a transition metal, which then initiates Fenton-type chemistry to generate reactive oxygen species similar to hydroxyl free radicals.
Research has shown that the MnSOD gene can express a splice isoform (isoMnSOD) during stress conditions that expresses a pro-oxidant form instead of the normal antioxidant activity of the normal MnSOD (Anziano, et al., Pediatrics Research, 47; 2000). IsoMnSOD is also described in WO 99/43697. MnSOD alternative splicing is inducible and depends on the deregulation of the normal MnSOD splicing pathway. Alternative splicing of the MnSOD RNA removes coding Exon 3, and fuses in-frame flanking Exons 2 and 4. The isoMnSOD protein is internally deleted for key alpha helical domain that serves in the parent MnSOD as a portal for the selective entry of superoxide anions into the MnSOD metal pocket. IsoMnSOD does not exhibit antioxidant, dismutase activity as the parent MnSOD, but exhibits in vitro a gain-of-function peroxidative activity that generates reactive oxygen free radicals from hydrogen peroxide (H2O2). In vivo, isoMnSOD initiates lipid peroxidation within the mitochondrial membrane and it causes modification of target proteins by oxidative stress markers such as the reactive lipid byproduct, 4-hydroxynonenal (HNE).
In addition, stress from internal factors (e.g. diseases) or by exogenous or external influences such as, for example, drugs, can impair a cell's and organism's viability. Although the pharmacological properties of drugs or potential drugs are well understood, companies still spend billions of dollars a year on candidates that fail during preclinical and clinical trials due to unforeseen drug toxicity. Current methods for predicting whether a drug will be toxic in an individual have not been particularly effective because there are few useful markers of drug-induced toxicity that would indicate whether a drug is worthwhile pursuing.
Additionally, there are numerous drugs that are used today whose effectiveness is diminished because of toxicity that is caused by inherent toxicity of the drugs, thereby limiting the useful dosage. Some of the toxicity that is caused by the drugs can be related to damage to mitochondrial contents due to altering and diminishing its antioxidant environment and, therefore, if the ancillary toxic event can be prevented it should enhance the effectiveness of the compounds.
In view of the above evidence, there is a need to identify modulators of isoMnSOD activity so that one can control the effects of isoMnSOD expression. There is also a need to identify compounds that can be used to reduce or prevent drug-induced toxicity. There are further needs for assays and methods that can be used to predict if a composition will cause drug-induced toxicity in an individual or a cell. The present invention helps to fulfill these needs as well as others.